JP2002503668A - How to synthesize GM3 - Google Patents

Abstract

The invention describes an improved method for making monosialoganglioside GM3 and its intermediates. Following reaction of a neuraminic acid donor and a lactose acceptor in the presence of an acid catalyst, the alpha and beta isomers of GM3 are formed. The alpha isomer is converted to a lactone, via action of a ring forming basic catalyst, which is then separable from the beta isomer. The lactone is then treated with a basic catalyst in the presence of an alcohol, to form GM3 or a GM3 intermediate.

Description

DETAILED DESCRIPTION OF THE INVENTION

RELATED APPLICATIONS This application is hereby incorporated by reference into this application, Feb. 1, 1996.
It is a continuation-in-part application of application number 08 / 620,580 filed on the 6th.

[0002] The present invention relates to a method for producing synthetic GM3. The compounds produced by the method of the present invention are as useful as all gangliosides and can also be used as starting materials for further synthetic reactions.

[0003] gangliosides of the invention is one of a class of molecules of glycolipids. Various gangliosides have been identified as prominent cell surface components of various transformed cells, including tumors of melanoma and other neuroectodermal origin. For example, Ritter and Livingstone (both of which are incorporated by reference into this application).
Livingston) et al . , Sem. Canc . Biol. , 2: 401-409 (1991) and Ottgen (O.
ettgen), VCH Verlags Gesellschaft (Weinheim Germany 1989).

[0004] Gangliosides are mono-glycosylated depending on the degree of glycosylation by sialic acid residues.
, Di-, tri- or polysialogangliosides. Abbreviations used to identify these molecules include "GM1", "GD3", "GT1"
Etc., where “G” represents ganglioside, “M”, “D”
"Or" T "and the like refer to the number of sialic acid residues, and a number or a number plus a letter (eg," GT1a ") refers to the binding pattern observed for the molecule. Lehninger, Biochemistry , pg. 294-296 (Worth Publishers
, 1981); Wiegandt, Glycolipids: New Comprehensive Bioche mistry (Neuberger et al., Ed., Elsevier, 1985), pp. 199-260.

The monosialoganglioside GM3 has the following structure: 2αNeuAc-3Galβ1-4GKβ1-ceramide

[0006] Gangliosides are widespread cell surface markers in transformed cells such as melanoma. This has made them attractive targets for cancer research. Livingston et al . , Proc. Natl. Acad. Sci. USA , 84: 2911-2915 (1987), the contents of which are incorporated herein by reference .
Describe the results of a vaccine-based study in which patients with melanoma were given whole cells showing high levels of GM2 as vaccine, pure GM2 or pure GM2 plus a bacterial adjuvant. . Livingston et al., J. Clin. Oncol ., 12 (5): 1036-1044 (1994) and Irie et al., U.S. Patent No. 4, which incorporates both contents into this application by reference. , 557, 931, which address the use of GM2 as a vaccine.

[0007] There are unique difficulties in the immunology of gangliosides, which are briefly mentioned here. First, while these molecules are widespread in transformed cells, they are also common in some normal cells, such as nerve cells. When gangliosides are administered to a patient, there is a risk that the resulting antibody response will damage normal cells. Indeed, certain autoimmune conditions, such as Guillain-Barre syndrome, are characterized by autoimmune antibodies that react with GM1 or GQ1b. See, for example, Yuki et al . , J. Exp. Med. , 178: 11771-1775 (1993); Aspinall et al., Infect & Immun., 6295: 2122-2125 (1994).

[0008] There is a further practical problem in that it is very difficult to secure a highly pure ganglioside in sufficient quantity for an immunization protocol. Currently, there is no practical synthesis method available. As a result, gangliosides are expressed in bovine brain (c
ranial) It is secured by purification from tissues such as tissues. Even under optimal conditions, the yield of pure gangliosides, including GM2 and GM3, is negligible. In addition, purification from mammalian tissues carries the risk of transmitting contaminants such as viruses and prion particles. Therefore, an alternative to existing methods for securing ganglioside-specific antibodies is highly desirable.

Because of the importance of gangliosides, it is desirable to develop a method for synthesizing pure gangliosides in high yield. The inventors of the present application have determined that pure GM in high yield
A new method for synthesizing 2 has been developed. Other methods of making synthetic GM2 are described in Hasegawa et al., J. Carbohydrate Chemistry , 11 (6): 699-714 (1992) and Sugimoto et al., Carbohydrate Research , 156: C1-C5 (1986). ). The present invention described herein extends the art in that the methods described herein are not suggested by these references.

[0010] If GM3 is used as a starting material, the synthesis of GM2 can be facilitated. However, for all the reasons mentioned above, it is difficult to obtain GM3 from the previously described raw materials. Therefore, it would be desirable to have available a method to promote mass production of GM3 that does not have the problems as indicated above. The invention relates, inter alia, to a method serving these purposes.

SUMMARY OF THE INVENTION The present invention relates to a method for synthesizing GM3 and GM3 intermediates, the result of which is:
Large quantities of the desired product are produced quickly. In one embodiment of this method, a neuraminic acid donor reacts with a lactose acceptor. By carrying out this reaction in the presence of an acid catalyst, a GM3 intermediate and a β-isomer of GM3 described in detail below are obtained. The β-isomer is an unwanted product of this reaction, but the desired GM3
It is difficult to quickly and efficiently separate the intermediate from the β-isomer. However, it has now been found that by reacting the desired GM3 intermediate in the presence of a basic catalyst, a lactone compound is formed and the lactone compound is easily separated from the β-isomer. The lactone can then react with a combination of a basic catalyst and an alcohol to form the desired GM3 or GM3 intermediate.
As a result, GM3 and its intermediates can be produced quickly and in gram quantities.

[0012] How this is achieved will be understood by reference to Example 8 and the following.

In the present invention, a GM3 intermediate is formed by reacting a neuraminic acid donor with a lactose donor using an acidic catalyst. Specifically, to form the desired compound, for example,

Embedded image When

Embedded image And react. Wherein R is a C1-C6 linear or branched alkyl, phenyl,
Or benzyl, optionally substituted at least once by a group comprising oxygen, nitrogen or sulfur. Preferably R is C1-C6 alkyl. Most preferably, it is straight-chain methyl, ethyl, propyl or butyl. R ¹ is benzyl, benzoyl, pivaloyl or acetyl.

As indicated above, this reaction not only forms GM3 or a GM3 intermediate, but also forms the β isomer. That is,

Embedded image and

Embedded image Where R is as previously indicated. These two classes of compounds, the desired compound and the β isomer, are not easily separable. However, they can be separated by adding a basic catalyst. Examples of the basic catalyst include 1,8-diazabicyclo (5.4.0) undes-7-ene (1
, 8-diazabicyclo (5.4.0) undec-7-ene) (hereinafter referred to as "DBU"), triethylamine, or other similar compounds. These compounds are catalytic in that they promote lactone ring formation and ring closure in the desired product when used alone, but promote the ring opening of the fusch ring when used with alcohols.

The following formula:

Embedded image After formation of the lactone represented by, the lactone can be easily separated from the β-isomer. The lactone is then reacted again with a basic catalyst in the presence of an alcohol represented by the formula ROH. Here, R may contain 1 to 10, preferably 1 to 6, carbon atoms, such as methyl or benzyl alcohol. In the presence of such an alcohol, the lactone ring opens and a GM3 intermediate is formed depending on the alcohol used.

This is illustrated in Example 2 below. If the lactone is available as a raw material, of course there is no need to make it and one can start by simply adding a basic catalyst and an alcohol.

Example 1 To prepare II ³ NeuAcGgOse ₃ Cer, herein referred to as GM3, an I, a neuraminic acid donor, and a compound II, a lactose acceptor, were prepared using the method of TJ Martin et al., Glycoconjugate. Prepared as described by J. 1993, supra . A solution of neuraminic acid donor (1 mmol) and lactose acceptor (1.5 mmol) in dry acetonitrile (5 mL) was cooled to −40 ° C. to obtain compound III, GM3. Catalyst (0.15 mmol) tin (II) trifluoromethanesulfonate under nitrogen atmosphere
(tin (II) trifluoromethanesulfonate) was added. After 1 hour, the solution was neutralized with triethylamine and evaporated in vacuo. The residue was purified by flash chromatography on silica gel using toluene-acetone (3: 1) as eluent to give compound III in 65% yield. For NMR data, see TJ Martin et al., Glycoconjugate J. 1993, supra .

Example 2 Example 1 illustrates a useful process for making milligram quantities of GM3. GM3
If mass production (i.e., gram quantities) is desired, better methods are needed.
Such a method is described below. In these methods, the same compounds used in Example 1 were combined. In the following structures, R ¹ may be benzyl, benzoyl, acetyl, pivaloyl, or the like. The compound is treated with an acid catalyst of the type previously described, Sn (II) trifluoromethanesulfonate (Sn (II) trifluorometha
nesulfonate). The products of this reaction are the GM3 intermediate and the beta isomer of GM3. That is,

Embedded image and

Embedded image It is. 1,8-diazabicyclo (5.4.0) undes-7 which is a basic catalyst
-Ene (1,8-diazabicyclo (5.4.0) undec-7-ene) is then added to the lactone, i.e.

Embedded image Is formed.

While it is very easy to separate the GM3 lactone from the GM3 β-isomer, it is very difficult to separate the α and β isomers.

Once the lactone has been separated from GM3, the lactone can be combined with an alcohol such as methyl alcohol and 1,8-diazabicyclo (5.4.0) undes-7-ene (1,8-diazabi).
Treatment with cyclo (5.4.0) undec-7-ene) gives the desired GM3 intermediate.

The details of this method are described below.

Example 3 Lactose acceptor described above wherein R ¹ is pivaloyl (200 mg, 0.23 mmol) in dry acetonitrile, and Sn (II) trifluoromethane (S
A solution of n (II) trifluoromethane) (25 mg, 0.06 mmol) was cooled to -40 <0> C. The neuraminic acid donor in dry acetonitrile (183 mg, 0
. 30 mmol) was added dropwise to this first solution under a nitrogen atmosphere.
This provided the desired GM3 and the β-isomer described above.

One hour after combining the two solutions, 90 ul (0.60 mmol) of 1
, 8-diazabicyclo (5.4.0) undes-7-ene (1,8-diazabicyclo (5.
4.0) undec-7-ene) was added and the resulting solution was stirred for 1 hour at room temperature.

This solution was then neutralized with an ion exchange resin, filtered and evaporated in vacuo. And the residue was purified by column chromatography. G mentioned earlier
A fast-moving spot corresponding to M3-lactone was seen, with a 60% yield (1
86 mg). On the other hand, spots traveling at a slower speed corresponding to the previously described β-GM3 were also seen, with a 6% yield (21 mg). When the lactone was examined for its NMR characteristics, it was confirmed that it was actually the desired lactone.

The lactone is separated from the column and taken up in a solution of dry methanol (200 mg, 0.17 mmol lactone, 5 ml methanol), cooled to −20 ° C. and less than 1 ul 1,8 -Diaza-bicyclo (5.4.0) undes-7-ene (1,8-diaza-bicyclo (5,4,0) undec-7-ene) was added. The solution was stirred at -20 ° C for 2 hours. Then the solution was neutralized with acetic acid and evaporated. Column chromatography provided 144 mg, or 70%, of the desired GM3 intermediate as product.

As indicated previously, this process results in the production of GM3 lactone. The GM3 lactone is easily separated from the β-isomer of GM3 and can be used to produce the desired GM3 intermediate. About 1/2 to get gram amount of GM3
Day time is needed. In contrast, when the former manufacturing method is used, about one week is required.

While the invention has been described with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of various aspects of the invention. Therefore,
It should be understood that many modifications can be made in the exemplary embodiments and other arrangements can be developed without departing from the spirit and scope of the invention.

[Procedure amendment]

[Submission date] September 25, 2000 (2000.9.25)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Claims

[Correction method] Change

[Correction contents]

[Claims]

Embedded image A compound of the following formula:

Embedded image Wherein R ¹ in each case is C ¹ -C 6 straight or branched alkyl, phenyl or benzyl, optionally substituted at least once, with a group containing oxygen, nitrogen or sulfur; Compound with the compound,

Embedded image Wherein R ¹ is benzyl, benzoyl, acetyl or pivaloyl,

Embedded image and

Embedded image Forming a mixture of, a step of adding a basic catalyst to the mixture to form a lactone of the following formula,

Embedded image separating the lactone from the β isomer and reacting the lactone with a basic catalyst and an alcohol to form the compound.

8. A method for making a compound of the formula:

Embedded image Wherein R ¹ is benzyl, benzoyl, pivaloyl or acetyl, comprising reacting a compound of the following formula with a basic catalyst and an alcohol to form said compound;

Embedded image Wherein R ¹ is as described above.

Wherein said alcohol is methyl alcohol or benzyl alcohol The method of claim 8.

Wherein said alcohol is methyl alcohol, The method of claim 9.

11. The method according to claim 11, wherein the basic catalyst is 1,8-diazabicyclo (5.4.0).
9. The method of claim 8 , wherein the method is undes-7-ene (1,8-diazabicyclo (5.4.0) undec-7-ene) or triethylamine.

12. The method of claim 11, wherein the basic catalyst is 1,8-diazabicyclo (5.4.0)
9. The method of claim 8 , which is 1,8-diazabicyclo (5.4.0) undec-7-ene.

────────────────────────────────────────────────── ─── Continuing from the front page (71) Applicant 605 THIRD AVENUE, NEW YORK, NEW YORK 10158, UNITED STATES OF AM ERICA (72) Inventor Castro-Palomino, Julio, Tse Deus-78434 Cons Stans Universi, Germany Tart Strasse 10 Em 725 (72) Inventor Ritter, Gerd New York 10021 New York York Avenue 1275 (72) Inventor Old, Lloyd, Jay United States New York 10105 New York Avenue of the Americas 1345 F-term (reference) 4C057 AA16 AA19 BB04 CC01 CC03 DD03 JJ03 JJ13 4H039 CA42 CA61 CG10 CG90

Claims (1)

Claims: 1. A method for preparing a compound of the formula: A compound of the following formula: Wherein R ¹ in each case is C ¹ -C 6 straight or branched alkyl, phenyl or benzyl, optionally substituted at least once, with a group containing oxygen, nitrogen or sulfur, Compounding with a compound, Wherein R ¹ is benzyl, benzoyl, acetyl or pivaloyl; And Forming a mixture of the following: adding a basic catalyst to the mixture to form a lactone of the following formula: separating the lactone from the β isomer and reacting the lactone with a basic catalyst and an alcohol to form the compound. 2. The method of claim 1, wherein said alcohol is methyl alcohol or benzyl alcohol. 3. The method of claim 2, wherein said alcohol is methyl alcohol. 4. The method according to claim 1, wherein the acid catalyst is tin (II) trifluoromethanesulfonate (tin).
(II) trifluoromethanesulfonate). 5. The method of claim 1, wherein the basic catalyst is 1,8-diazabicyclo (5.4.0) undes-7-ene (1,8-diazabicyclo (5.4.0) undec-7-ene) or triethylamine. Item 1. The method of Item 1. 6. The basic catalyst is 1,8-diazabicyclo (5.4.0) undec-7-ene, which is 1,8-diazabicyclo (5.4.0) undec-7-ene. the method of. 7. The method according to claim 1, further comprising a step of separating the lactone from the β isomer by column chromatography. 9. A method for making a compound of the formula: Wherein R ¹ is benzyl, benzoyl, pivaloyl or acetyl, comprising reacting a compound of the following formula with a basic catalyst and an alcohol to form said compound: Wherein R ¹ is as described above. 10. The method of claim 9, wherein said alcohol is methyl alcohol or benzyl alcohol. 11. The method of claim 10, wherein said alcohol is methyl alcohol. 12. The method according to claim 1, wherein the basic catalyst is 1,8-diazabicyclo (5.4.0).
10. The method of claim 9 which is undes-7-ene (1,8-diazabicyclo (5.4.0) undec-7-ene) or triethylamine. 13. The method according to claim 1, wherein the basic catalyst is 1,8-diazabicyclo (5.4.0).
10. The method of claim 9, which is undes-7-ene (1,8-diazabicyclo (5.4.0) undec-7-ene).